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J. Biol. Chem., Vol. 280, Issue 13, 12676-12682, April 1, 2005

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Tumor Necrosis Factor- Up-regulates the Expression of 1,4-Galactosyltransferase I in Primary Human Endothelial Cells by mRNA Stabilization^*

Juan Jesús García-Vallejo, Willem van Dijk, Irma van Die, and Sonja I. Gringhuis

From the Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Centre, 1007 MB Amsterdam, The Netherlands

Received for publication, September 2, 2004 , and in revised form, January 3, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
During the course of an inflammatory response, the pro-inflammatory cytokine tumor necrosis factor- (TNF) triggers endothelial cells to increase the expression levels of adhesion molecules that are pivotal for the rolling, adhesion, and transmigration of leukocytes over the endothelial cell wall. Here we show that TNF, in addition, has a regulatory function in the biosynthesis of proper carbohydrate molecules on endothelial cells that constitute ligands for adhesion molecules on leukocytes. Our data show that TNF induced an increase in the expression of 1,4-galactosyltransferase-1 (4GalT-1) in primary human umbilical vein endothelial cells in a time- and concentration-dependent manner. The 4GalT-1 mRNA up-regulation correlated with an increase in the Golgi expression and catalytic activity of the enzyme. Furthermore, an enhanced incorporation of galactose was observed in newly synthesized glycoproteins. Analysis of the molecular mechanism behind the up-regulation of 4GalT-1 showed that the increase in mRNA levels is due to an enhanced stability of the transcripts. These data strongly demonstrate that TNF modulates the glycosylation of endothelial cells by a mechanism that directly enhances the stability of 4GalT-1 mRNA transcripts.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The cytokine TNF¹ is released during the inflammatory response by activated macrophages (acute inflammation) or lymphocytes (chronic inflammation) situated at the site of injury. It activates endothelial cells to expose adhesion molecules that are necessary for the rolling, adhesion, and transmigration of leukocytes through the endothelial cell wall (1). Glycans play important roles in the recruitment of leukocytes because they are involved in many of the leukocyte-endothelial interactions. It has been reported previously that the glycan biosynthesis changes during an immune response (2). Accordingly, in recent preliminary data, we observed that TNF induces changes in the expression levels of several glycosyltransferase genes² in primary HUVECs, of which up-regulation of the mRNA of 4GalT-1 was the most prominent one.

To date, 4GalT-1 (E.C. 2.4.1.22 [EC] /38) is one of the best-studied glycosyltransferases. Although several aspects of its regulation and function have been extensively studied (3–5), there are still many uncharacterized features. 4GalT-1 is constitutively expressed in all tissues, with the exception of the brain (6), as a Golgi-resident protein (7). It catalyzes the transfer of galactose from the activated sugar donor, UDP-galactose, to oligosaccharides carrying terminal N-acetyl-glucosamine. This reaction results in the formation of 4-N-acetyllactosamine (Gal1,4-GlcNAc) or poly-4-N-acetyllactosamine sequences, also known as type 2 chains, that are abundantly present in N- and O-linked glycans as well as in glycolipids (8). Thus, 4GalT-1 plays an important role in the synthesis of the backbone structure of important carbohydrate epitopes involved in leukocyte-endothelial cell interactions, such as selectin ligands, and also galectin and siglec ligands (9). Additionally, in the lactating mammary gland, -lactalbumin converts 4GalT-1 to lactose synthase. Interaction of 4GalT-1 with this cofactor greatly lowers the K_m of the enzyme for glucose, so that glucose can be effectively utilized as acceptor substrate at physiological concentrations (10, 11).

Although 4GalT-1 is ubiquitously expressed in human tissues, the levels of expression vary and are subject to different stimuli, as in lactation (12, 13), T-lymphocyte activation and differentiation (14, 15), and activation of monocytic cells (16). In the case of the lactating mammary gland, the regulatory mechanism involves two steps: an initial 10-fold increase in mRNA levels is achieved by transcriptional regulation (12, 17), and an additional 5-fold increase is obtained by translational control (13). During mid- and late pregnancy, the expression of lactating gland-specific transcription factors induces a change in the transcription initiation site, leading to the expression of a shorter transcript. These shorter mRNAs contain less stable secondary structures in the 5'-UTR and, as a result, are translated more efficiently than the longer and highly structured transcript of the non-lactating gland. However, the regulatory mechanisms in tissues other than the lactating mammary gland differ completely and are not yet fully elucidated. The regulatory mechanism in activated HL60 cells, in contrast with lymphocytes, seems to involve post-transcriptional control at the level of mRNA stability (14, 16). A further indication that the expression of 4GalT-1 might be regulated at the post-transcriptional level is found in the short half-life of 80 min of its mRNA in mice (18), similar to other labile mRNAs that are also regulated at the level of mRNA stability (19).

In the present study, we set out to investigate the mechanism involved in the TNF-mediated up-regulation of 4GalT-1 in primary HUVECs and to explore the effect on the activity and subcellular localization of the enzyme. Our data show that4GalT-1 is up-regulated in a time- and concentration-dependent manner in response to TNF stimulation due to an increase in the stability of the mRNA transcript. Furthermore, the 4GalT-1 mRNA up-regulation correlated with an increase in the Golgi-restricted expression of the enzyme and an augmented in vitro 4GalT-1 activity and resulted in an enhanced galactose incorporation in newly synthesized glycoproteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Cultures and Reagents—HUVECs were isolated from five healthy donors by a modification of the method of Jaffe et al. (20). The cells were cultured in M199 supplemented with 100 units/ml penicillin-streptomycin, 10% human serum, 10% newborn calf serum, 5 units/ml heparine (Leo Pharmaceutical Products), and 10 ng/ml basic fibroblast growth factor (Sigma) on 1% gelatin (Fluka)-coated 6-well plates (Costar). When confluence was reached, cells were trypsinized (0.18% trypsin, 10 mM EDTA) and plated again to one-third of their density. All experiments were performed 2 passages after isolation. Cells were serum-starved for 2 h and exposed to 100 units/ml TNF (Strahtmann Biotech) for 6 h or as otherwise indicated in the text.

The human cervical epithelial carcinoma cell line HeLa Tet-Off (Clontech), which is stably transfected to continuously express the tetracycline-controlled transactivator, was cultured in high-glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (Clontech) supplemented with 2 mM L-glutamine, 4.5 g/liter D-glucose, 100 units/ml penicillin, and 100 units/ml streptomycin. Cells were serum-starved for 2 h prior to experiments.

Plasmids—The 3'-UTR of the 4GalT-1 gene (GenBank^TM NM_001497 [GenBank] , bases 1269 – end) was cloned into the BglII site of the pTetBBB plasmid (a kind gift from Dr. A. Shyu, Houston, TX) (21), which contains the rabbit -globin gene, to generate pTetBBB/4GalT1–3'UTR to allow the analysis of the effect of the 4GalT-1 3'-UTR on the stability of the very stable rabbit -globin mRNA. When this plasmid is transfected into cell lines expressing the tetracycline-controlled transactivator, such as the HeLa Tet-Off cell line, then a chimeric mRNA is continuously transcribed. The four ATTTA sequences within the 4GalT-1 3'-UTR (AUUUA in mRNA) were changed to ACGTA using the QuikChange II XL site-directed mutagenesis kit (Stratagene) as indicated in Fig. 5A, thus generating pTetBBB/4GalT1–3'UTR(mAU1), pTetBBB/4GalT1–3'UTR(mAU2), pTetBBB/4GalT1–3'UTR(mAU3), and pTetBBB/4GalT1–3'UTR(mAU4).

View larger version (34K): [in this window] [in a new window]   FIG. 5. The TNF-induced increase in mRNA stabilization involves AU2. A, HUVECs were grown to confluence in 24-well plates, serum-starved for 2 h, and incubated for 6 h in the presence or absence of 100 units/ml TNF. Actinomycin D (10 µg/ml) was added to the cell cultures, and cells were lysed for mRNA isolation as described under "Experimental Procedures" at the selected time points (0, 20, 40, 60, 90, 120, and 240 min). Results are shown as the average ± S.D. of the remaining 4GalT-1 transcripts in five independent experiments. B, HUVECs were grown to confluence in 24-well plates, serum-starved for 2 h, and incubated for 0, 10, 30, 60, or 360 min in the presence of 100 units/ml TNF. After the time points mentioned, actinomycin D (10 µg/ml) was added to the cell cultures, and cells were lysed for mRNA isolation 0, 20, 40, 60, 90, 120, and 240 min after actinomycin D addition. C, subsequently, 4GalT-1 half-life was determined as described in A. Results are shown as the average ± S. D. of the 4GalT-1 half-life in five independent experiments.

mRNA Half-life Determination—HUVECs were stimulated as previously indicated, and 10 µg/ml actinomycin D (Sigma) was added to block transcription. After stimulation of transfected HeLa Tet-Off, 1 µg/ml doxycycline (Clontech) was added to block tetracycline-controlled transactivator-dependent transcription of chimeric mRNA. Lysates for mRNA isolation were taken 0, 30, 60, 120, 180, and 240 min after the addition of either actinomycin D or doxycycline.

Isolation of RNA and cDNA Synthesis—mRNA was specifically isolated by capturing of poly(A⁺) RNA in streptavidin-coated tubes with a mRNA Capture kit (Roche Applied Science), and cDNA was synthesized with the reverse transcription system kit (Promega) following the manufacturer's guidelines. Cells (2 x10⁵ cells/well) were washed twice with ice-cold PBS and harvested with 200 µl of lysis buffer. Lysates were incubated with biotin-labeled oligo(dT)₂₀ for 5 min at 37 °C, and then 50 µl of the mix were transferred to streptavidin-coated tubes and incubated for 5 min at 37 °C. After washing three times with 250 µl of washing buffer, 30 µl of the reverse transcription mix (5 mM MgCl₂,1x reverse transcription buffer, 1 mM deoxynucleotide triphosphate, 0.4 unit of recombinant RNasin ribonuclease inhibitor, 0.4 unit of avian myeloblastosis virus reverse transcriptase, and 0.5 µg of random hexamers in 30 µl of nuclease-free water) were added to the tubes and incubated for 10 min at room temperature followed by a 45-min incubation at 42 °C. To inactivate avian myeloblastosis virus reverse transcriptase and separate mRNA from the streptavidin-biotin complex, samples were heated at 99 °C for 5 min, transferred to microcentrifuge tubes, and incubated in ice for 5 min. Then, they were diluted 1:2 in nuclease-free water and stored at –20 °C until analysis.

Real-time PCR—Oligonucleotides (Table I) have been designed by using Primer Express 2.0 computer software (Applied Biosystems). All oligonucleotides were provided by Invitrogen. Oligonucleotide specificity was computer tested (BLAST; National Center for Biotechnology Information) by homology search with the human genome and specifically with all the known galactosyltransferases (CLUSTALW; European Molecular Biology Laboratory), and later confirmed by dissociation curve analysis and resolving the PCR products in agarose electrophoresis. The efficiency (22) of the oligonucleotides was determined using the computer program LinReg (23) and resulted in an average of 90%. PCRs were performed in an ABI 7900HT sequence detection system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's guidelines. The fluorescence monitoring occurred at the end of each cycle. Additionally, dissociation curve analysis was performed at the end of every run, showing in all cases one single peak at the Tm (melting temperature of the amplicon) predicted by the Primer Express 2.0 software. The Ct (cycle at the threshold) value is defined as the number of PCR cycles where the fluorescence signal exceeds the detection threshold value, which is fixed above 10 times the S.D. of the fluorescence during the first 15 cycles and typically corresponds to 0.2 relative fluorescence unit. This threshold is set constant throughout the study and corresponds to the log linear range of the amplification curve. The normalized amount of target (24) reflects the relative amount of target transcripts with respect to the expression of the endogenous reference gene. In this study, the endogenous reference gene chosen was glyceraldehyde 3-phosphate dehydrogenase, based on previous results (25).

Flow Cytometry—Cells were washed twice in cold PBS and resuspended in PBS. Subsequently, cells were fixed with 3% paraformaldehyde/PBS (10 min, room temperature), centrifuged, and treated with 20 mM glycine/PBS (10 min, room temperature) in order to quench free aldehyde groups. Subsequently, cells were permeabilized with 0.1% saponin/PBS for 30 min, incubated for 30 min at room temperature with 25 µl of the primary antibody (GT2/36/118) diluted in 0.1% saponin/1% bovine serum albumin-PBS, washed twice with 0.1% saponin/PBS, and incubated for 30 min at room temperature with secondary antibody (PE-rabbit anti-mouse) diluted in 0.1% saponin/1% bovine serum albumin-PBS. After the second incubation, cells were washed twice with 0.1% saponin/PBS and resuspended in a final volume of 100 µl of 0.1% saponin/1% bovine serum albumin-PBS for analysis in the FACSCalibur (BD Biosciences). Cells were analyzed for immunofluorescence by collecting data for 10⁴ cells/histogram. Corresponding negative controls were performed by using a secondary antibody alone.

Galactosyltransferase Assay—Galactosyltransferase assays were performed as described previously (27) by incubating cell lysates in 100 mM sodium cacodylate buffer, pH 7.2, 20 mM MnCl₂, 4 mM ATP, 1% Triton X-100, 0.5 mM UDP-[³H]galactose (specific activity, 10 Ci/mol), and 30 mM N-acetyl-glucosamine. After incubation at 37 °C, the product was isolated by ion-exchange using Dowex 1x8-200 resin (Sigma) and separated in HPAEC-PAD (high pH anion-exchange chromatography with pulsed amperometric detection), using Gal-1,3-GlcNAc and Gal-1,4-GlcNAc as standards. Radioactivity incorporated into the product was determined in a 1900TR liquid scintillation analyzer (Packard).

Metabolic Labeling and Autoradiography—HUVECs were grown to confluence in T175 flasks, serum-starved for 2 h, and incubated for 10 h with 5 µCi/ml [³H]galactose (American Radiolabeled Chemicals) in the presence or absence of 100 units/ml TNF (Strahtmann Biotech). Cells were washed and resuspended in PBS and lysed in Laemmli sample buffer. The cell lysate was subjected to SDS-PAGE in a 10% polyacrylamide gel according to Laemmli (28) and blotted onto a polyvinylidene difluoride membrane (Millipore). The membrane was exposed for autoradiography in a tritium screen (Amersham Biosciences) and analyzed with a STORM^TM 860 system (Amersham Biosciences).

Statistics—Results are shown as the average ± S.D. of five independent measurements. Statistical significance was evaluated using the Kruskal-Wallis test in the SPSS 11.0 software (license number AZVU-7061649). Computer analysis of the 4GalT-1 gene and mRNA sequences was performed using TRANSFAC (29) and the CpGplot application at European Molecular Biology Laboratory-European Bioinformatics Institute (www.ebi.ac.uk/Tools/sequence.html).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF Up-regulates 4GalT-1 mRNA Expression in HUVECs in a Time- and Concentration-dependent Manner—One of the functions of TNF during the inflammatory response is to activate endothelial cells and to prompt them to produce adhesion molecules, which are necessary for the recruitment of leukocytes to the injured tissue. Preliminary observations showed that TNF modulates the expression of several glycosylation-related genes.² Among the genes up-regulated by TNF in endothelial cells were 4GalT-1, -5, and -6, whereas no effect was apparent in other 3- or 4-galactosyltransferases. The expression of 4GalT-1 was considerably higher than that of other 4-galactosyltransferases. Expression of 3-galactosyltransferases was more than 50-fold less abundant than 4GalT-1 expression. Hence, we decided to focus on 4GalT-1. To establish the kinetics of the TNF-mediated up-regulation of 4GalT-1 mRNA levels, confluent monolayers of primary HUVECs were incubated in the presence of increasing concentrations of TNF and for different times. Prior to this study, glyceraldehyde 3-phosphate dehydrogenase was determined as the optimal endogenous reference gene for normalization in the experimental conditions used in the present work (25). The expression of E-selectin was determined as a positive control for the activation of HUVECs. Low concentrations of TNF (20 units/ml) were ineffective in up-regulating both E-selectin and 4GalT-1, whereas concentrations between 50 and 250 units/ml produced an almost linear increase in the expression, reaching plateau values at higher concentrations (Fig. 1A). For analysis of the time dependence of this effect, HUVECs were incubated with culture medium alone or with medium containing 100 units/ml TNF, and the relative abundance of E-selectin and 4GalT-1 transcripts was determined by quantitative real-time PCR. The activation of HUVECs by TNF resulted in a >2-fold increase in the expression of 4GalT-1 at 6 h after the addition of the cytokine (Fig. 1B) and a >500-fold increase in the E-selectin mRNA. After this time point, mRNA levels slowly decreased, returning to the basal levels, although a small degree of up-regulation was still visible after 12 h of incubation.

View larger version (18K): [in this window] [in a new window]   FIG. 1. The treatment of HUVECs with TNF induces an up-regulation of 4GalT-1 in a concentration- and time-dependent manner. A, HUVECs were grown to confluence in 24 well-plates, serum-starved for 2 h, and incubated for 6 h in the presence or absence of 20, 50, 100, 200, or 500 units/ml TNF. Cells were lysed, and the relative abundance of E-selectin and 4GalT-1 transcripts was determined by real-time PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous reference gene as described under "Experimental Procedures." B, HUVECs were serum-starved for 2 h and incubated in the presence or absence of 100 units/ml TNF for 0, 2, 4, 6, 8, 10, and 12 h. At the different time points, cells were lysed, and the relative abundance of 4GalT-1 was determined as indicated above. Results in A and B represent the average ± S.D. of five independent experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 2. The localization of 4GalT-1 is restricted to the Golgi and up-regulated by TNF. HUVECs were grown to confluence in GlassTek II slides, serum-starved for 2 h, and incubated for 8 h in the absence (A) or presence (B) of 100 units/ml TNF. After fixation, the cells were permeabilized; stained with GT2/36/118, phalloidin, and Hoechst; and analyzed for immunofluorescence in a Nikon Eclipse E800 microscope. C, HUVECs were grown to confluence in T25 flasks, serum-starved for 2 h; incubated for 8 h in the presence or absence of 100 units/ml TNF; mechanically detached and subsequently fixated, permeabilized, and stained with GT2/36/118; and analyzed for immunofluorescence using a FACSCalibur. Results are representative of three separate experiments.

View larger version (68K): [in this window] [in a new window]   FIG. 3. TNF-induced increase in galactose incorporation in newly synthesized glycoproteins in HUVECs. HUVECs were grown to confluence in T175 flasks and incubated with 5 mCi of (C6)-[3H]galactose in the presence or absence of 100 units/ml TNF. After 24 h, cells were lysed in CHAPS, and identical amounts of protein were subjected to SDS-PAGE, followed by transfer onto polyvinylidene difluoride membrane and autoradiography using a tritium screen. The fold change values represent the ratio of TNF-treated to non-treated radioactivity for the indicated bands. Results are representative of three separate experiments.

In the lactating mammary gland, there is a change in the transcription initiation site that results in 4GalT-1 transcripts with a lower GC content in the 5'-UTR. The resulting less structured transcripts are translated more efficiency (13). However, this is unlikely to occur in endothelial cells, so we directed our attention to the 3'-UTR. Interestingly, the 3'-UTR of 4GalT-1 presents four AUUUA sequences (AREs), which are often associated with the regulation of mRNA stability (19). To test whether mRNA stabilization is involved in the up-regulation of 4GalT-1, the half-life of 4GalT-1 transcripts was determined in resting and TNF-treated HUVECs using the transcription inhibitor actinomycin D. The results clearly showed that TNF stimulation induced an increase in the half-life from 147 ± 24 to 288 ± 96 min (p < 0.01) after TNF treatment (Fig. 4A). These data imply that the 2-fold increase in mRNA levels as shown in Fig. 1 is due to an enhanced mRNA stability. Next, the kinetics of this TNF-mediated mRNA stabilization were investigated by determining the half-life of 4GalT-1 at different time points after TNF stimulation. The results clearly indicate that after 30 min, the half-life had already increased significantly (p < 0.01) (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   FIG. 4. The up-regulation of 4GalT-1 by TNF is a post-transcriptional phenomenon involving mRNA stabilization. A, HUVECs were grown to confluence in 24-well plates, serum-starved for 2 h, and incubated for 6 h in the presence or absence of 100 units/ml TNF. Actinomycin D (10 µg/ml) was added to the cell cultures, and cells were lysed for mRNA isolation as described under "Experimental Procedures" at the selected time points (0, 20, 40, 60, 90, 120, and 240 min). Results are shown as the average ± S.D. of the remaining 4GalT-1 transcripts in five independent experiments. B, HUVECs were grown to confluence in 24-well plates, serum-starved for 2 h, and incubated for 0, 10, 30, 60, or 360 min in the presence of 100 units/ml TNF. After the time points mentioned, actinomycin D (10 µg/ml) was added to the cell cultures, and cells were lysed for mRNA isolation 0, 20, 40, 60, 90, 120, and 240 min after actinomycin D addition. Subsequently, 4GalT-1 half-life was determined as described in A. Results are shown as the average ± S.D. of the 4GalT-1 half-life in five independent experiments.

As previously mentioned, the 3'-UTR of 4GalT-1 contains four AREs, termed AU1 through AU4 for simplicity, with AU1 being closest to the stop codon of 4GalT-1 (Fig. 5A). In order to study the individual contribution of each ARE to the TNF-induced mRNA stabilization of 4GalT-1, every ARE was consecutively mutated in the Globin-4GalT1–3'UTR construct (Fig. 5A). The elimination of binding sites for AU-binding proteins by individually mutating the AUUUA sequences of AU1 through AU4 to ACGUA only affected the half-life of the chimeric mRNA when AU2 was mutated (Fig. 5B). Chimeric mRNA harboring the mutated AU2 (Globin-4GalT1-mAU2) showed an enhanced half-life (257 ± 55 min, p < 0.01), similar to endogenous 4GalT-1 mRNA in TNF-stimulated HUVECs (Fig. 4A). Furthermore, TNF did not induce an additional stabilization of chimeric Globin-4GalT1-mAU2 mRNA (Fig. 5C). These results indicate that the stability of the 4GalT-1 mRNA is modulated solely through one ARE within its 3'-UTR (AU2). Finally, these results suggest that in resting cells a destabilizing factor is bound to AU2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Although 4GalT-1 has long been considered a housekeeping gene, evidence is accumulating showing that its expression is regulated and involved in important functions (3). In this report, we provide new evidence showing that TNF up-regulates the expression of 4GalT-1 in endothelial cells by a post-transcriptional mechanism involving the control of mRNA stability. Furthermore, the time- and concentration-dependent increase in mRNA levels is accompanied by an increase in the Golgi localization of the enzyme and the enzyme activity, suggesting a role for 4GalT-1 in the changes occurring in the glycosylation of endothelial cells during inflammation.

4GalT-1 mRNA expression is regulated by TNF in a time- and concentration-dependent manner, indicative of a direct effect of TNF signaling. The involvement of delayed regulatory systems, such as the synthesis of intermediate regulatory molecules or the effect of secondary cytokines secreted by HUVECs under the influence of TNF (32), is excluded by the short time needed to reach a maximum effect as well as by the observation that the activation by TNF appears to be saturable.

Computational analysis of the 5'-flanking region of the 4GalT-1 gene revealed the presence of a CpG island and a high frequency of binding sites for the transcription factor Sp1. CpG islands have been associated with a low degree of methylation and, consequently, with an "active" state of transcription (33), whereas Sp1 is a transcription factor generally associated with the transcription of so-called "housekeeping genes" (34). Because no binding sites for transcription factors related to TNF signaling pathways were found in the 5'-flanking region of the 4GalT-1 gene, we hypothesized that the transcription of 4GalT-1 is set at a basal level, and consequently, it is likely that other mechanisms are involved in the regulation of gene expression of 4GalT-1 by TNF. Indeed, we could observe an increase in the half-life of 4GalT-1 mRNA after the treatment of HUVECs with TNF. This finding is in agreement with the presence of AUUUA sequences in the 3'-UTR of 4GalT-1. Furthermore, the rapid effect of TNF in increasing the 4GalT-1 mRNA half-life (30 min) suggests the involvement of a rapid activation system. The analysis of the 4GalT-1 3'-UTR in the well-known Tet-Off system (21) clearly demonstrated that AU2 is the ARE responsible for the TNF-induced increase in mRNA stability. AUUUA sequences are also known as AREs and are potential binding sites for mRNA stability-modulating proteins (19). Several RNA-binding proteins, such as tristetraproline, K homology-type splicing regulatory protein, AUF1, and HuR, have been shown to form stable complexes with AREs and influence in this way the degradation rate, either positively or negatively, of mRNA (19). In particular, tristetraproline and HuR should be considered as a priori candidates for the modulation of the 4GalT-1 mRNA stability because their binding is regulated by the p38 (35) and phosphatidylinositol 3-kinase (36) signaling pathways, which are also activated by TNF (37). Furthermore, tristetraproline has long been known as a destabilizing factor (38). The identities of the 4GalT-1 ARE-binding proteins and how they are affected by TNF signaling pathways are currently under investigation. It is still unclear why only AU2 is involved in the stabilization of the 4GalT-1 mRNA. It has previously been suggested that the function of AU-binding proteins might be dependent on the density of ARE in a particular sequence and the formation of mRNA secondary structures (39, 40).

The TNF-induced stabilization of the 4GalT-1 transcripts in HUVECs results in an increase in the expression of the enzyme in the Golgi, the in vitro enzyme activity in cell lysates, and the incorporation of galactose into newly synthesized glycoproteins. Immunostainings of 4GalT-1 in HUVECs showed a juxtanuclear pattern compatible with localization in the Golgi apparatus and are in agreement with previous reports (41). Although ectopic localization of 4GalT-1 has been described previously (3, 42, 43) and associated with adhesive, lectin-like properties of the enzyme in the absence of the sugar donor UDP-galactose, we could not demonstrate any plasma membrane localization after sequential analysis of consecutive planes ranging from the basolateral to the apical membrane (data not shown). We conclude from these data that the up-regulation of 4GalT-1 transcripts results in a functional increase in 4-galactosylation mediated by the increase in the Golgi expression of the enzyme. Indeed, TNF-treated HUVECs demonstrated an increase in the incorporation of galactose in proteins with a molecular mass between 45 and 100 kDa, which coincides with the molecular mass of numerous integrins and adhesion molecules (intercellular adhesion molecule-1, intercellular adhesion molecule-2, and vascular cell adhesion molecule-1) that are expected to act as scaffolds for the presentation of carbohydrate ligands for selectins, galectins, or siglecs. Interestingly, it has been shown that 6'-sialyllactosamine, the ligand for the B-cell molecule CD22, is up-regulated in TNF-treated HUVECs, which was has been attributed to an increase in the expression of ST6Gal I (44, 45). However, the TNF-induced up-regulation of 4GalT-1 described in the present study can be expected to contribute to the change in glycan synthesis. This might be of special importance in the migration of B cells to the inflamed joints in rheumatoid arthritis, where B cells are found in high numbers and partly contribute to the progression of the disease (46). Accordingly, TNF blockade therapy reduces the numbers of synovial CD22+ B cells in rheumatoid arthritis patients (47). This may involve blocking of the TNF-dependent up-regulation of 4GalT-1 and ST6Gal I. The TNF-dependent up-regulation of 4GalT-1 might also be critical in the expression of 6'sulfo-3'sialyl-3fucosyl-lactosamine, the L-selectin ligand, in activated endothelial cells (48). The up-regulation of L-selectin ligands is the main mechanism for the migration of monocytes and lymphocytes in chronic inflammation, as has been demonstrated in several models, such as contact hypersensitivity (49), Arthus reaction (50), Sjögren's syndrome (51), or ulcerative colitis (52). The further dissection of the mechanisms involved in regulating the expression of 4GalT-1 is important to increase our understanding of the biosynthesis of complex-type glycans that constitute ligands for adhesion molecules during inflammation, which will enable the design of more specific therapies to fight chronic inflammatory diseases.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY864848 [GenBank] .

To whom correspondence should be addressed: Glycoimmunology Group, Dept. of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, P. O. Box 7057, 1007 MB Amsterdam, The Netherlands. Tel.: 31-20-4448156; Fax: 31-20-4448144; E-mail: jj.garciavallejo{at}vumc.nl.

¹ The abbreviations used are: TNF, tumor necrosis factor-; UTR, untranslated region; ARE, AU-rich element; 4GalT-1, 1,4-galactosyltransferase 1; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

² J. J. García-Vallejo, W. van Dijk, B. van het Hof, I. van Die, M. A. Engelse, V. W. M. van Hinsbergh, and S. I. Gringhuis, submitted for publication.

	ACKNOWLEDGMENTS

 
We thank Dr. C. Grün, Ing. B. Bruyneel, and Ing. W. Schiphorst for excellent assistance with the HPAEC-PAD. We also thank Dr. E. G. Berger for the kind gift of the GT2/36/118 antibody, Dr. A. B. Shyu for the kind gift of plasmid pTetBBB, Dr. H. de Vries for the kind gift of phalloidin-rhodamine, Dr. R. Beelen for help in providing umbilical cords, and Dr. G. Kraal for helpful discussions and critical reading of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Collins, T., Read, M. A., Neish, A. S., Whitley, M. Z., Thanos, D., and Maniatis, T. (1995) FASEB J. 9, 899–909[Abstract]
2. Lowe, J. B. (2003) Curr. Opin. Cell Biol. 15, 531–538[CrossRef][Medline] [Order article via Infotrieve]
3. Berger, E. G., and Rohrer, J. (2003) Biochimie (Paris) 85, 261–274
4. Shaper, J. H., Charron, M., Lo, N. W., Scocca, J. R., and Shaper, N. L. (2000) in Carbohydrates in Chemistry and Biology: A Comprehensive Handbook (Ernst, B., Hart, G., and Sinay, P., eds) pp. 175–196, Wiley/VCH Press, New York
5. Shaper, J. H., and Shaper, N. L. (2001) in Handbook of Glycosyltransferases and Their Related Genes (Taniguchi, N., and Fukuda, M., eds) pp. 11–19, Springer, Tokyo
6. Lo, N. W., Shaper, J. H., Pevsner, J., and Shaper, N. L. (1998) Glycobiology 8, 517–526[Abstract/Free Full Text]
7. Roth, J., and Berger, E. G. (1982) J. Cell Biol. 93, 223–229[Abstract/Free Full Text]
8. Kotani, N., Asano, M., Iwakura, Y., and Takasaki, S. (2001) Biochem. J. 357, 827–834[CrossRef][Medline] [Order article via Infotrieve]
9. Asano, M., Nakae, S., Kotani, N., Shirafuji, N., Nambu, A., Hashimoto, N., Kawashima, H., Hirose, M., Miyasaka, M., Takasaki, S., and Iwakura, Y. (2003) Blood 102, 1678–1685[Abstract/Free Full Text]
10. Bell, J. E., Beyer, T. A., and Hill, R. L. (1976) J. Biol. Chem. 251, 3003–3013[Abstract/Free Full Text]
11. Hill, R. L., and Brew, K. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 411–490[Medline] [Order article via Infotrieve]
12. Rajput, B., Shaper, N. L., and Shaper, J. H. (1996) J. Biol. Chem. 271, 5131–5142[Abstract/Free Full Text]
13. Charron, M., Shaper, J. H., and Shaper, N. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14805–14810[Abstract/Free Full Text]
14. Lemaire, S., Derappe, C., Pasqualetto, V., Mrkoci, K., Berger, E. G., Aubery, M., and Neel, D. (1998) Glycoconj. J. 15, 161–168[CrossRef][Medline] [Order article via Infotrieve]
15. Nakayama, F., Teraki, Y., Kudo, T., Togayachi, A., Iwasaki, H., Tamatani, T., Nishihara, S., Mizukawa, Y., Shiohara, T., and Narimatsu, H. (2000) J. Investig. Dermatol. 115, 299–306[CrossRef][Medline] [Order article via Infotrieve]
16. Pasqualetto, V., Lemaire, S., Neel, D., Aubery, M., Berger, E. G., and Derappe, C. (2000) Carbohydr. Res. 328, 301–305[CrossRef][Medline] [Order article via Infotrieve]
17. Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348–14359[Abstract/Free Full Text]
18. Scocca, J. R., Charron, M., Shaper, N. L., and Shaper, J. H. (2003) Biochimie (Paris) 85, 403–407
19. Bevilacqua, A., Ceriani, M. C., Capaccioli, S., and Nicolin, A. (2003) J. Cell. Physiol. 195, 356–372[CrossRef][Medline] [Order article via Infotrieve]
20. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973) J. Clin. Investig. 52, 2745–2756
21. Xu, N., Loflin, P., Chen, C., and Shyu, A. (1998) Nucleic Acids Res. 26, 558–565[Abstract/Free Full Text]
22. Pfaffl, M. W. (2001) Nucleic Acids Res. 29, E45
23. Ramakers, C., Ruijter, J. M., Lekanne Deprez, R. H., and Moorman, A. F. M. (2003) Neurosci. Lett. 339, 62–66[CrossRef][Medline] [Order article via Infotrieve]
24. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402–408[CrossRef][Medline] [Order article via Infotrieve]
25. Garcia-Vallejo, J. J., Van het Hof, B., Robben, J., Van Wijk, J. A. E., Van Die, I., Joziasse, D. H., and van Dijk, W. (2004) Anal. Biochem. 329, 293–299[CrossRef][Medline] [Order article via Infotrieve]
26. Berger, E. G., Aegerter, E., Mandel, T., and Hauri, H. P. (1986) Carbohydr. Res. 149, 23–33[CrossRef][Medline] [Order article via Infotrieve]
27. Bakker, H., Van Tetering, A., Agterberg, M., Smit, A., Van den Eijnden, D., and Van Die, I. (1997) J. Biol. Chem. 272, 18580–18585[Abstract/Free Full Text]
28. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
29. Wingender, E. (2000) Trends Glycosci. Glycotechnol. 12, 255–264
30. Van Die, I., Van Tetering, A., Schiphorst, W. E. C. M., Sato, T., Furukawa, K., and Van den Eijnden, D. H. (1999) FEBS Lett. 450, 52–565[CrossRef][Medline] [Order article via Infotrieve]
31. Takizawa, M., Nomura, T., Wakisaka, E., Yoshizuka, N., Aoki, J., Arai, H., Inoue, K., Hattori, M., and Matsuo, N. (1999) Biochim. Biophys. Acta 1438, 301–304[Medline] [Order article via Infotrieve]
32. Mantovani, A., Garlanda, C., Introna, M., and Vecchi, A. (1998) Transplant. Proc. 30, 4239–4243[CrossRef][Medline] [Order article via Infotrieve]
33. Antequera, F. (2003) Cell. Mol. Life Sci. 60, 1647–1658[CrossRef][Medline] [Order article via Infotrieve]
34. Bouwman, P., and Philipsen, S. (2002) Mol. Cell. Endocrinol. 195, 27–38[CrossRef][Medline] [Order article via Infotrieve]
35. Zhu, W., Brauchle, M. A., Di Padova, F., Gram, H., New, L., Ono, K., Downey, J. S., and Han, J. (2001) Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L499–L508[Abstract/Free Full Text]
36. Ming, X. F., Stoecklin, G., Lu, M., Looser, R., and Moroni, C. (2001) Mol. Cell. Biol. 21, 5778–5789[Abstract/Free Full Text]
37. MacEwan, D. (2002) Cell. Signal. 14, 477–492[CrossRef][Medline] [Order article via Infotrieve]
38. Blackshear, P. J. (2002) Biochem. Soc. Trans. 30, 945–952[CrossRef][Medline] [Order article via Infotrieve]
39. Meisner, N. C., Hackermüller, J., Uhl, A., Aszódi, A., Jaritz, M., and Auer, M. (2004) Chembiochem. 5, 1432–1447[CrossRef][Medline] [Order article via Infotrieve]
40. Zhang, T., Kruys, V., Huez, G., and Gueydan, C. (2002) Biochem. Soc. Trans. 30, 952–958[CrossRef][Medline] [Order article via Infotrieve]
41. Schnyder-Candrian, S., Borsig, L., Moser, R., and Berger, E. G. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8369–8374[Abstract/Free Full Text]
42. Berger, E. G. (2002) Glycobiology 12, 29R–36R[Abstract/Free Full Text]
43. Shur, B. D., Evans, S., and Lu, Q. (1998) Glycoconj. J. 15, 537–548[CrossRef][Medline] [Order article via Infotrieve]
44. Hanasaki, K., Varki, A., and Powell, L. D. (1995) J. Biol. Chem. 270, 7533–7542[Abstract/Free Full Text]
45. Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637–10643[Abstract/Free Full Text]
46. Smeets, T. J. M., Barg, E. C., Kraan, M. C., Smith, M. D., Breedveld, F. C., and Tak, P. P. (2003) Ann. Rheum. Dis. 62, 635–638[Abstract/Free Full Text]
47. Taylor, P. C., Peters, A. M., Paleolog, E., Chapman, P. T., Elliott, M. J., McCloskey, R., Feldmann, M., and Maini, R. N. (2000) Arthritis Rheum. 43, 38–47[CrossRef][Medline] [Order article via Infotrieve]
48. Spertini, O., Luscinskas, F. W., Gimbrone, M. A., Jr., and Tedder, T. F. (1992) J. Exp. Med. 175, 1789–1792[Abstract/Free Full Text]
49. Hirata, T., Furie, B. C., and Furie, B. (2002) J. Immunol. 169, 4307–4313[Abstract/Free Full Text]
50. Kaburagi, Y., Hasegawa, M., Nagaoka, T., Shimada, Y., Hamaguchi, Y., Komura, K., Saito, E., Yanaba, K., Takehara, K., Kadono, T., Steeber, D. A., Tedder, T. F., and Sato, S. (2002) J. Immunol. 168, 2970–2978[Abstract/Free Full Text]
51. Mikulowska-Mennis, A., Xu, B., Berberian, J. M., and Michie, S. A. (2001) Am. J. Pathol. 159, 671–681[Abstract/Free Full Text]
52. Renkonen, J., Tynninen, O., Hayry, P., Paavonen, T., and Renkonen, R. (2002) Am. J. Pathol. 161, 543–550[Abstract/Free Full Text]

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